Biosensor, magnetic molecule measurement method, and measurement object measuring method

ABSTRACT

A biosensor not requiring washing of an unbonded label molecular by analyzing an object to be measured such as an antigen, an antibody, a DNA, and an RNA through magnetic field sensing. The biosensor is small in size, low in price, and high in sensing accuracy. Semiconductor Hall devices are arrayed two-dimensionally on the bottoms of recesses in the surface of a sensor chip to which bonded is a magnetic molecule with which a magnetic particle is labeled so as to sense the magnetic field produced along the sensor surface of the sensor chip. The surface area of each semiconductor Hall device is less than the maximum cross section of the magnetic molecule, and the intervals of the arrayed semiconductor Hall devices are larger than the diameter of the magnetic Hall molecule. Thus, the analysis accuracy is enhanced. An interconnection is used commonly to the semiconductor Hall devices, thereby reducing the size of the sensor.

TECHNICAL FIELD

The present invention relates to a biosensor, a method for measuringmagnetic molecules and a method for measuring objects to be measured,and more particularly to a biosensor, a method for measuring magneticmolecules and a method for measuring objects to be measured that areused for analyzing objects to be measured by measuring the amount ofmagnetic molecules.

BACKGROUND ART

In recent years clinical diagnosis and detection as well as geneticanalysis is being carried out by immunological techniques orhybridization in which specific binding between specific pairs ofmolecules, such as binding of an antigen to its antibody, is utilized todetect antigens, antibodies, DNA (DeoxyribonucleicAcid) molecules, RNA(RibonucleicAcid) molecules and the like.

Particularly in solid phase binding assay, a method is used thatutilizes magnetic particles for detection. A schematic diagram of theconventional solid phase assay using magnetic particles is shown in FIG.17.

As shown in the figure, the assay is performed using a solid phase 91,molecular receptors 61 that capture an object to be measured 62, amagnetic particle 51, and molecular receptors 63 which detect the objectto be measured 62, to thereby assay the object to be measured 62.

The solid phase 91 has a solid phase surface that contacts with a samplesolution, and the molecular receptors 61 are immobilized on the solidphase surface. A polystyrene bead, a wall surface of a reaction vessel,a substrate surface or the like is used for the solid phase.

As the molecular receptors 61 and the molecular receptors 63, a moleculethat binds specifically to the object to be measured 62 that is presentin the sample solution is used. The object to be measured 62 may be anantigen, antibody, DNA molecule, RNA molecule or the like.

The magnetic particle 51 is a labeling material having magnetization.Detecting a magnetic field produced by the magnetization of the magneticparticle makes it possible to determine the amount of the magneticparticle 51 that is in a state to be described hereinafter, and thus thepresence or concentration of an object to be measured in the samplesolution can be identified. In addition to the magnetic particle 51, asubstance that emits a detectable signal, such as a radioactivesubstance, fluorophore, chemiluminophore, enzyme or the like, may beused as a label. Examples of known assay methods using these labelsinclude Enzyme Immunoassay (EIA) which utilizes the reaction between anantigen and its antibody, or Chemiluminescence (CL) methods such as astrict Chemiluminescence Immunoassay (CLIA) in which a chemiluminescentcompound is used as a labeling compound for immunoassay, orChemiluminescence Enzyme Immunoassay (CLEIA) in which enzyme activity isdetected at high sensitivity using a chemiluminescent compound in thedetection system.

The molecular receptors 63 that are previously bound to the magneticparticle 51 are antibodies that bind specifically to the object to bemeasured 62 that is previously bound to the magnetic particle 51.

In the assay illustrated in the figure, first, a sample solutioncontaining the object to be measured 62 is introduced onto the solidphase 91 on which the molecular receptors 61 were immobilizedbeforehand, whereby the object to be measured 62 specifically binds tothe molecular receptor 61. Other substances contained in the samplesolution float in the sample solution without binding to the solid phase91. Then, the magnetic particle 51 on which the molecular receptors 63are immobilized is introduced into the sample solution. Alternatively,the magnetic particle 51 on which the molecular receptors 63 areimmobilized may be introduced into the sample solution at the same timeas the object to be measured 62. Thereby, the molecular receptor 63binds specifically to the object to be measured 62 that is boundspecifically to the molecular receptor 61 immobilized on the solidphase. The magnetic particle 51 having the molecular receptors 63immobilized thereon is referred to as a “magnetic molecule”. Then, amagnetic field produced by the magnetic particle is detected, wherebythe amount of magnetic particles 51 bound to the surface of the solidphase 91 is determined. Thus, it is possible to determine theconcentration or position of the object to be measured 62 bound to thesurface of the solid phase 91. With respect to detection of the magneticfield, detection methods using magnetoresistive elements arranged in anarray are disclosed in U.S. Pat. No. 5,981,297 and International PatentPublication WO 97/45740.

In addition to the sandwich assay method described above in which anobject to be measured binds specifically to a molecular receptor and adifferent molecule label then binds specifically to the object to bemeasured, examples of other assay methods utilizing the above labelsinclude a competitive assay in which an object to be measured andanother molecule label competitively bind to a molecular receptor.

In the conventional methods, a signal such as fluorescence or the likefrom a label is detected by an apparatus, such as an optical detectionapparatus, that is capable of detecting the signal. In these methods, itis necessary to capture only the signal from the label of a moleculethat is specifically bound to a binding molecule immobilized on a solidphase surface. However, in the case of optical detection, if an unboundlabeled molecule is present, the signal from this label may also becaptured and thus an accurate assay cannot be conducted.

Accordingly, it is necessary to completely wash away unbound labelingmolecules. Further, since it is necessary for an optical detectionapparatus to detect a faint light signal, it is difficult to produce aminiturized or low cost detection apparatus.

In the method for detection by magnetoresistive elements using magneticparticles as labels as disclosed in the above U.S. Pat. No. 5,981,297,it is not necessary to wash away any unbound labeling molecules.However, a detection chip on which magnetoresistive elements arearranged in an array requires the switching circuits in order toindependently extract the signal of individual elements. Electricalwiring is then required respectively from each of the elements in thearray to the switching circuits. Consequently, one problem therewith isthat as the number of elements increases the wiring becomes morecomplicated and the area occupied by the wiring also increases, and thusminiaturization is difficult.

In the aforementioned International Patent Publication WO 97/45740, acircuit for detection of magnetic particles comprises a bridge circuitcomposed of magnetoresistive elements and transistors functioning asswitching elements. However, since magnetoresistive elements requiremagnetic material, after fabricating a part of the circuit containingtransistors by a standard integrated circuit production process, it isthen necessary to conduct processes for forming and processing amagnetic thin film.

It is an object of the present invention to provide a biosensor thateliminate the need of washing away of unbound labeling molecules byanalyzing an object to be measured such as an antigen, antibody, DNAmolecule or RNA molecule through detection of a magnetic field, in whichthe biosensor is small in size, low in price, and high in sensingaccuracy, as well as a method for measuring a magnetic molecule and amethod for measuring an object to be measured.

DISCLOSURE OF THE INVENTION

According to the present invention there is provided a biosensor whichassays an object to be measured by means of a magnetic sensor composedof detector elements for detecting a magnetic field produced by boundmagnetic molecules, the detector elements being arrangedtwo-dimensionally in X rows and Y columns (X and Y are natural numbers,the same applies hereunder) and the assay of the object to be measuredbeing conducted by measuring the amount of the above magnetic molecules,characterized in that the biosensor comprises a signal processing meanswhich determines the amount of the bound magnetic molecules by comparingthe intensities of magnetic fields of different regions along the sensorsurface of the magnetic sensor, and assay of the object to be measuredis conducted based on the determined amount of magnetic molecules.

The present invention further provides the above biosensor characterizedin that the detector element comprises a semiconductor Hall device.

The present invention further provides the above biosensor characterizedin that a reference region to which the above magnetic molecules areincapable of binding is provided on the sensor surface of the abovemagnetic sensor, and the above signal processing means conductscomparison using an intensity of a magnetic field of the referenceregion as a reference.

As used herein, the term “mutually different regions” refers to, forexample, comparing the respective intensities of magnetic fieldsdetected by adjacent Hall devices. Alternatively, a region is providedas a reference, and the intensity of a magnetic field detected in thatregion may be used as a reference for comparison with the intensity of amagnetic field detected in another arbitrary region.

The present invention further provides the above biosensor characterizedin that surface treatment is performed for the above reference regionsuch that molecular receptors that bind with the magnetic molecules areincapable of immobilizing thereto.

The present invention further provides the above biosensor characterizedin that it further comprises a selection means for selecting individualdetector elements arranged in the above X rows and Y columns andextracting an output thereof.

The present invention further provides the above biosensor characterizedin that the above magnetic sensor, the above selection means and asignal amplification circuit that amplifies an output signal of theabove detector element are formed on one chip.

The present invention further provides the above biosensor characterizedin that the size of each detection space in which detection of amagnetic field by the above detector elements is possible is equal tothe size of approximately one molecule of the magnetic molecules to bebound.

Thereby, the number of magnetic molecules detected by a detector elementis limited to one, and thus variations in measurement values caused bydetection of a plurality of magnetic molecules can be controlled. Thisenables enhancement of the analysis accuracy.

The present invention further provides the above biosensor characterizedin that in the above magnetic sensor, the detector elements are arrangedat intervals such that two detector elements next to each other detectmutually different magnetic molecules.

Thus, it is possible to inhibit interference such as detection of thesame magnetic molecule by adjacent detector elements.

The present invention still further provides the above biosensorcharacterized in that the above adjacent detector elements are arrangedadjacent to each other at an interval that is equal to or greater thanthe diameter of the above magnetic molecules.

Thus, it is possible to inhibit interference such as detection of thesame magnetic molecule by adjacent semiconductor Hall devices.

The present invention also provides the above biosensor characterized inthat the sensor surface of the above magnetic sensor is subject tosurface treatment for selectively immobilizing in a specified regionmolecular receptors that bind to the above magnetic molecules.

The present invention further provides the above biosensor characterizedin that recesses of a size corresponding to the size of the abovemagnetic molecules are provided on the sensor surface of the abovemagnetic sensor, and that molecular receptors that bind to the magneticmolecules are provided only in the recesses on the sensor surface.

The present invention still further provides the above biosensorcharacterized in that a gold thin film is formed in the specified regionon the sensor surface of the magnetic sensor, and the above molecularreceptors that have an end modified by a thiol group are selectivelyimmobilized thereon.

The present invention also provides the above biosensor characterized inthat the biosensor comprises a first magnetic field generation meansarranged in a position facing the sensor surface of the above magneticsensor that generates a magnetic field that is applied to the sensorsurface of the above magnetic sensor.

The present invention further provides the above biosensor characterizedin that it comprises means whereby the above first magnetic fieldgeneration means intermittently generates a magnetic field.

The present invention still further provides the above biosensorcharacterized in that it comprises a detector circuit which, when theabove first magnetic field generation means generates a magnetic fieldat a constant frequency, extracts only a frequency componentcorresponding to the magnetic field from an output signal of the abovedetector element.

The present invention further provides the above biosensor characterizedin that the biosensor comprises a second magnetic field generation meansarranged on the backside of a sensor surface of the above magneticsensor for generating a magnetic field that is applied to the sensorsurface of the above magnetic sensor.

The present invention further provides the above biosensor characterizedin that the above second magnetic field generation means comprises meansfor intermittently generating a magnetic field.

The present invention further provides the above biosensor characterizedin that the when the above second magnetic field generation meanscomprises a detector circuit for generating a magnetic field at aconstant frequency, extracting only a frequency component correspondingto the magnetic field from an output signal of the above detectorelement.

The present invention further provides the above biosensor characterizedin that the above magnetic sensor is arranged such that the sensorsurface of the magnetic sensor faces in a direction in whichgravitational force acts.

The present invention still further provides the above biosensorcharacterized in that the above semiconductor Hall device has a pair ofcurrent terminals, a gate electrode controlling current flowing betweenthe current terminals, and a pair of output terminals arranged such thatcurrent flows roughly perpendicular to current flowing between thecurrent terminals.

The present invention further provides the above biosensor characterizedin that, in the above case, the above gate electrode is connected to agate electrode wire that is common to the semiconductor Hall devicesarranged in the same column, the above pair of current terminals areconnected to a pair of current terminal wires that are common to thesemiconductor Hall devices arranged in the same row, the above pair ofoutput terminals are connected to a pair of output terminal wires thatare common to the semiconductor Hall devices arranged in the same row,and the above selection means extracts an output signal of thesemiconductor Hall device arranged in an arbitrary position by selectingthe gate electrode wire, pair of current terminal wires, and pair ofoutput terminal wires.

By providing interconnections that are common for each column and eachrow, respectively, selection of a semiconductor Hall device in anarbitrary position can be simply performed and, moreover, the number ofinterconnections can be reduced. Thus, production of a magnetic sensoraccording to an object to be measured sample is simplified, andminiaturization of a magnetic sensor is enabled.

According to a further aspect of the present invention there is provideda method for measuring magnetic molecules in which a magnetic sensorcomposed of detector elements for detecting a magnetic field produced bybound magnetic molecules, in which the detector elements are arrangedtwo-dimensionally in X rows and Y columns (X and Y are natural numbers,the same applies hereunder), is used to determine an amount of themagnetic molecules, characterized in that the method comprises:

-   -   a measurement step for acquiring intensities of magnetic fields        of mutually different regions of a sensor surface of the        magnetic sensor; and    -   a determination step for determining the amount of the bound        magnetic molecules by comparing the intensities of the magnetic        fields of the mutually different regions obtained in the        measurement step.

The present invention further provides the above method for measuringmagnetic molecules characterized in that in the above determinationstep, comparison is carried out taking as a reference the intensity of amagnetic field of a reference region to which magnetic molecules areincapable of binding that was obtained in the above measurement step.

According to another aspect of the present invention there is provided amethod for measuring magnetic molecules in which a magnetic sensorcomposed of detector elements for detecting a magnetic field produced bybound magnetic molecules, in which the detector elements are arrangedtwo-dimensionally in X rows and Y columns (X and Y are natural numbers,the same applies hereunder), is used to determine the amount of themagnetic molecules, characterized in that the method comprises:

-   -   a pre-binding measurement step for acquiring intensities of        magnetic fields prior to binding of the magnetic molecules;    -   a post-binding measurement step for acquiring intensities of        magnetic fields after binding of the magnetic molecules; and    -   a determination step for determining the amount of the bound        magnetic molecules by comparing the intensities of the magnetic        fields prior to binding with the intensities of the magnetic        fields after binding.

The present invention further provides the above method for measuringmagnetic molecules characterized in that it further comprises an offsetvalue acquisition step for acquiring an offset value that is output fromthe detector element.

The present invention still further provides the above method formeasuring magnetic molecules characterized in that in the abovemeasurement step, by means of a magnetic field applied at a constantfrequency to the sensor surface, an output signal of a detector elementis obtained that includes a signal output at a frequency correspondingto the magnetic field; and in the above determination step, comparisonis conducted using a value obtained after removing an offset valueincluded as a direct current component by extracting only a frequencycomponent corresponding to the magnetic field from the output signal ofa detector element obtained in the above measurement step.

The present invention also provides the above method for measuringmagnetic molecules characterized in that it further comprises a bindingacceleration step for generating a magnetic field for bringing the abovemagnetic molecules close to the sensor surface by a magnetic fieldgeneration means at the time of introducing the magnetic molecules tothe sensor surface.

The present invention further provides the above method for measuringmagnetic molecules characterized in that in the above bindingacceleration step a magnetic field is applied of an intensity such thatthe magnetization of the above magnetic molecules becomes saturated, andin the above step of acquiring the intensity of a magnetic field, themagnetic field is applied of an intensity such that the magnetization ofthe magnetic molecules does not become saturated.

Here, a step for acquiring the intensity of a magnetic field is theabove measurement step, the above post-binding measurement step or theabove pre-binding measurement step or the like.

The present invention further provides the above method for measuringmagnetic molecules, characterized in that the method further comprises astirring step for stirring magnetic molecules, after introducing theabove magnetic molecules to the sensor surface, by alternately producingmagnetic fields by means of a first magnetic field generation meansarranged in a position facing the sensor surface and a second magneticfield generation means arranged on the backside of the sensor surface.

The present invention also provides the above method for measuringmagnetic molecules, characterized in that in the above stirring step amagnetic field is applied of an intensity such that magnetization of themagnetic molecules becomes saturated, and in a step for acquiring theintensity of a magnetic field a magnetic field is applied of anintensity such that magnetization of the magnetic molecules does notbecome saturated.

Here, a step for acquiring the intensity of a magnetic field is theabove measurement step, the above post-binding measurement step or theabove pre-binding measurement step or the like.

According to a further aspect of the present invention there is provideda method for measuring objects to be measured using a biosensoraccording to any of claims 1 to 20, characterized in that molecules thatbind specifically to the objects to be measured for binding are used asthe above magnetic molecules, the method comprising the steps of:

-   -   determining the amount of the magnetic molecules bound        specifically to objects to be measured using a biosensor; and    -   determining the amount of objects to be measured based on the        amount of the magnetic molecules.

According to a still further aspect of the present invention there isprovided a method for measuring objects to be measured using a biosensoraccording to any of claims 1 to 20, characterized in that molecules thatare reversibly exchangeable for the objects to be measured for bindingare used as the magnetic molecules, the method comprising the steps of:

-   -   determining an amount of the magnetic molecules bound in place        of the objects to be measured using a biosensor; and    -   determining the amount of the objects to be measured based on        the amount of the magnetic molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view schematically showing a part of the biosensor of thepresent invention;

FIG. 2 is a view illustrating the detection principles of a biosensoraccording to a first and second embodiment herein;

FIG. 3A is a sectional view of a sensor chip, and FIGS. 3B and 3C aresectional views of a sensor chip that was subjected to treatment forenhancing the sensitivity of a semiconductor Hall device;

FIG. 4A is a view showing the configuration of a semiconductor Halldevice according to the present invention, FIG. 4B is a sectional viewalong an alternate long and short dash line denoted by the referencecharacter “a” in FIG. 4A, and FIG. 4C is a sectional view along analternate long and short dash line denoted by the reference character“b” in FIG. 4A;

FIG. 5 is a view illustrating a method for selecting Hall devices in anarray according to the present invention;

FIG. 6 is a block diagram illustrating a circuit of a biosensoraccording to the present invention;

FIG. 7 is a flowchart illustrating the circuit operation of the entirebiosensor according to the first embodiment herein;

FIG. 8 is a flowchart illustrating the circuit operation of the entirebiosensor according to the second embodiment herein;

FIG. 9 is a view illustrating the detection principles of the biosensoraccording to the third embodiment herein;

FIG. 10 is a view illustrating the states of a magnetic field applied tomagnetic molecules by a coil in the third embodiment herein;

FIG. 11 is a flowchart illustrating the circuit operation of the entirebiosensor according to the third embodiment herein;

FIG. 12 is a view showing the state of disposition of Hall devices inthe third embodiment herein;

FIG. 13 is a table showing values used for comparison of output valuesin the third embodiment herein;

FIG. 14 is a view schematically showing a cross section of the sensorchip used in the fourth embodiment herein;

FIG. 15 is a flowchart illustrating the circuit operation of the entirebiosensor according to the fourth embodiment herein;

FIG. 16 is a table showing values for output signals prior to bindingand output signals after binding; and

FIG. 17 is a schematic diagram illustrating a conventional method ofsolid phase assay using a magnetic particle.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, embodiments of the present invention will be described referring tothe drawings. In each of the drawings referred to in the followingdescription, parts that are the same in other drawings are denoted usingthe same symbol.

Embodiment 1

FIG. 1 is a schematic diagram showing a part of a sensor chip comprisingthe biosensor of the present invention. The composition of a sensor chip1 includes semiconductor Hall devices as detector elements and a signalprocessing circuit of the semiconductor Hall devices. The sensor chip 1is produced as described hereunder.

The sensor chip 1 is formed on a silicon substrate 11 by well-known CMOS(complementary metal oxide semiconductor) processes. Semiconductor Halldevices are formed at the bottom of recesses 13 in the surface of thesensor chip 1. Input and output of each of the semiconductor Halldevices is conducted through a gate electrode 30 and a metal wire 4.

After forming semiconductor Hall devices and a signal processing circuiton the silicon substrate 11 by CMOS processes, molecular receptors suchas antigens, antibodies, DNA molecules or RNA molecules are immobilizedon the surface of the sensor chip 1 treated by a silane coupling agentor the like.

Then, a sample solution is dropped onto the surface of the sensor chip1, and objects to be measured such as antigens, antibodies, DNAmolecules or RNA molecules are bound to the molecular receptors on thesurface of the sensor chip 1. Thereafter, magnetic particles on whichantigens, antibodies, DNA molecules or RNA molecules or the like arebound are introduced onto the surface of the sensor chip 1, and arespecifically bound on semiconductor Hall devices to which objects to bemeasured are specifically binding.

The semiconductor Hall device preferably has a detection space of a sizethat can accommodate one of the specifically binding magnetic molecules.More specifically, in this embodiment, a semiconductor Hall devicepreferably has a space capable of detecting a magnetic field produced bya magnetic molecule binding to a molecular receptor immobilized on thesurface of the sensor chip 11. As used herein, the term “magneticmolecule” refers to a molecule that has magnetization. In thisembodiment, a magnetic molecule is a molecule labeled with a magneticparticle having a molecular receptor attached thereto. Alternatively,the molecule itself has magnetization and can be detected by asemiconductor Hall device. In this embodiment, the surface area of asemiconductor Hall device 2 is of a size equal to that of the maximumcross-section area of a magnetic molecule.

Thus, it is possible to limit the number of magnetic molecules 5 thatare present in a detection space to one. Therefore, when conductingmeasurement by detecting the presence or absence of one of the magneticmolecules 5 using one of the semiconductor Hall devices 2, it ispossible to prevent detection of two or more of the magnetic molecules 5by one semiconductor Hall device, thereby enabling accurate measurement.The present invention is not limited to a measurement method that iscarried out by one semiconductor Hall device detecting the presence orabsence of one magnetic molecule. More specifically, by making the sizeof the surface area of the semiconductor Hall device 2 equivalent to themaximum cross-section area of a plurality of magnetic molecules, onesemiconductor Hall device may detect the presence of a plurality ofmagnetic molecules.

Further, as shown in FIG. 1, a similar effect can be obtained byproviding recesses 13 in the detection spaces on the surface of thesensor chip 11, and making the area of the recesses 13 in correspondenceto the size of the magnetic molecules 5 used in each measurement. Forexample, the recesses 13 may be provided at a size that is smaller thanthe maximum cross-section area of the magnetic molecules 5 describedabove. Molecular receptors are then provided only in the recesses 13.Thus, the quantity of the magnetic molecules 5 that are capable ofbinding to molecular receptors can be controlled by limiting the numberof molecular receptors in the detection space regardless of the size ofthe semiconductor Hall devices. This configuration can be used when themagnetic molecules 5 are extremely minute in comparison to the size ofthe semiconductor Hall devices and it is not possible to reduce the sizeof the detection spaces. In this embodiment, the recesses 13 areconstructed by providing the metal wire 4 on the surface where thesemiconductor Hall devices are provided, however, for example, therecesses 13 may also be formed by etching after flattening the surfaceof the sensor chip 1. Molecular receptors can be immobilized only in therecesses 13 by first immobilizing molecular receptors to the sensor chip1 and then wiping the sensor chip surface 11.

The present invention is not limited to a case of providingsemiconductor Hall devices on the bottom of recesses as shown in FIG. 1,and semiconductor Hall devices may be provided such that the sensor chipsurface is flat. However, by forming semiconductor Hall devices on thebottom of the recesses 13 in the sensor chip surface, in addition to theaforementioned effects, the following effects can also be obtained. Thatis, interference caused by magnetic molecules present at the boundary ofa detection space of a semiconductor Hall device can be prevented byadjusting the binding conditions for magnetic molecules in regionsdetected by semiconductor Hall devices, or alternatively, the conditionsfor reaction between molecular receptors and magnetic molecules can bemade uniform by immobilizing molecular receptors only in regions whichare under the same conditions.

In this embodiment, semiconductor Hall devices are provided in the formof a two-dimensional array. In the sensor chip in this case,semiconductor Hall devices are arranged at intervals that are providedsuch that two adjacent semiconductor Hall devices detect mutuallydifferent magnetic molecules. Thus, it is possible to preventinterference among the individual semiconductor Hall devices. A similareffect can also be obtained by providing recesses corresponding tomutually different semiconductor Hall devices at the aforementionedintervals as explained above. As shown in FIG. 1, in this embodiment therecesses 13 corresponding to the respective semiconductor Hall devicesare provided at an interval W that is larger than a diameter R of themagnetic molecule 5.

The following method may be used as a method for immobilizing molecularreceptors such as antigens, antibodies, DNA molecules or RNA moleculeson the surface of the sensor chip 1. Semiconductor Hall devices and asignal processing circuit are formed on the silicon substrate 11 by CMOSprocesses, and then a gold thin film is formed on the sensor chipsurface. In order to improve adherence between the gold thin film andthe sensor chip surface, a thin film of Cr, Ni, or Ti is preferablyformed between the gold thin film and the surface of the sensor chip 1as an adhesion layer.

After formation of the gold thin film, molecular receptors having endsmodified with a thiol group are immobilized on the surface of the goldthin film. In this case, after formation of the gold thin film, it isalso possible to immobilize thiol compounds on the surface of the goldthin film and then immobilize the molecular receptors thereon.

Further, a gold thin film may be formed only at positions correspondingto the locations of the semiconductor Hall devices shown by the recesses13 in FIG. 1. Since thiol groups selectively bind with the gold thinfilm, it is possible to immobilize molecular receptors on only specificregions on the surface of the sensor chip 1.

A pattern of a gold thin film to be formed at specific positions can beformed by a so-called “lift-off method” in which a pattern is firstformed by a photoresist, and subsequently a Ti thin film as an adhesionlayer and a gold thin film are formed by sputtering, and the photoresistis then removed.

Next, the detection principles of a biosensor according to the presentinvention will be explained using FIG. 2. FIG. 2 is a view thatschematically shows a cross section of the vicinity of the semiconductorHall device 2 on the sensor chip 1. Molecular receptors 61 composed ofantibodies are immobilized on the surface of the semiconductor Halldevice 2. An object to be measured 62 binds specifically to themolecular receptor 61, and a magnetic particle 51 further binds to theobject to be measured 62 through specific binding between the object tobe measured 62 and a molecular receptor 63 composed of an antibody. Themagnetic particle 51 and the molecular receptors 63 bind to each otherto form the magnetic molecule 5.

An upper coil CU (first magnetic field generation means) is arranged ina position facing the surface of the sensor chip 1. In a state where amagnetic molecule is bound to the surface of the sensor chip 1 asdescribed above, a current is passed through the upper coil CU togenerate a magnetic field. In place of a coil, a permanent magnet or thelike may be used. In FIG. 2, a magnetic flux B is formed in thedirection indicated by an arrow Z, and the figure shows that thedirection thereof is perpendicular to the surface of the semiconductorHall device. Since the magnetic flux B is concentrated by the magneticparticle 51, the magnetic flux density at the semiconductor Hall device2 is increased in comparison to a case where the magnetic particle 51 isnot present. Further, because the magnetic field is applied from anupper coil, the magnetic flux density increases in accordance with thedistance away from the surface of the sensor chip 1. Therefore, floatingmagnetic molecules 5 that are not bound to the surface of the sensorchip 1 are attracted upward and do not influence the magnetic fluxdensity that is detected by the semiconductor Hall device 2. Since theoutput voltage of the semiconductor Hall device 2 is in proportion tothe magnetic flux density, whether or not the magnetic molecule 5 isbound on the semiconductor Hall device 2 can be determined by means ofthe output voltage.

When detecting the presence of a plurality of magnetic molecules withone semiconductor Hall device 2, since the incremental amount of theincrease in magnetic flux density due to concentration by a magneticparticle is dependent on the number of magnetic particles, the number ofmagnetic molecules bound on one semiconductor Hall device 2 can also bedetected.

In this embodiment a lower coil CD (second magnetic field generationmeans) is arranged on the backside of the sensor chip 1. The lower coilCD is not provided for the purpose of detecting magnetic molecules, butis provided to generate a magnetic field for bringing magnetic moleculesclose to the surface of the sensor chip 1. In place of a coil, apermanent magnet or the like maybe used. Upon introduction of magneticmolecules onto the sensor chip 1, a current is passed through the lowercoil CD to generate a magnetic field. Magnetic molecules are attractedto the surface of the sensor chip 1 by the magnetic field that is formedsuch that the magnetic flux density decreases in accordance with anincrease in the distance from the surface of the sensor chip 1, and thusthe time taken for binding of magnetic particles to the surface of thesensor chip 1 is reduced.

FIGS. 3A to 3C show sectional views of a sensor chip that is subjectedto treatment for enhancing the sensitivity of a semiconductor Halldevice. The sensor chip 1 is formed by CMOS processes as describedabove, in which the distance from the gate electrode 30 to the chipsurface is normally several μm. A sensing surface of the semiconductorHall device with respect to magnetic flux is formed in the interfacebetween the gate electrode 30 and a p-well region 36. Because thesensitivity of a semiconductor Hall device with respect to a magneticmolecule is in inverse proportion to the distance thereof from thesensing surface, it is preferable that the thickness of an insulatinglayer 12 formed on the gate electrode 30 be thin.

Accordingly, after the sensor chip 1 is produced by standard CMOSprocesses, as shown in FIG. 3B, the insulating layer 12 is removed byetching to a degree such that only the Hall device region is not exposedby the gate electrode 30. Further, as shown in FIG. 3C, a metal layer 37comprising aluminium or the like may be provided beforehand on the gateelectrode 30 as an etching stop layer.

FIG. 2 shows a magnetic molecule 5 that is used in a method formeasuring an object to be measured using a biosensor that assays anobject to be measured by detecting a magnetic field produced by amagnetic molecule. In this method, a molecule that binds specifically tothe object to be measured 62 is used as the magnetic molecule 5, theamount of the magnetic molecule 5 binding specifically to the object tobe measured 62 is determined using a biosensor, and the amount of theobject to be measured 62 is determined based on the amount of themagnetic molecule 5. However, the biosensor of the present invention isnot limited to use only when measuring an object to be measured bydetecting a magnetic molecule in this manner. For example, as themagnetic molecule, a molecule may be used that is labeled with amagnetic particle and binds with the surface of a magnetic sensor incompetition with an object to be measured. In this case, the amount ofmagnetic molecules binding to the surface in place of the object to bemeasured can be determined using a biosensor, and the amount of thecompeting object to be measured can be determined based on the amount ofthe magnetic molecule.

The configuration of the semiconductor Hall device of the presentinvention will be described referring to FIGS. 4A to 4C.

FIG. 4A shows a top view of the semiconductor Hall device 2, FIG. 4Bshows a sectional view along the alternate long and short dash linedenoted by the reference character “a” in FIG. 4A, and FIG. 4C shows asectional view along the alternate long and short dash line denoted bythe reference character “b” in FIG. 4A. The composition of thesemiconductor Hall device 2 includes the gate electrode 30, a sourceelectrode 31, a drain electrode 32, output electrodes 33 and 34, and aninsulating layer 35. The semiconductor Hall device 2 is formed in thep-well region 36. The configuration is the same as that of an n-typeMOSFET with the exception of the output electrodes. Metal wires to therespective electrodes have been omitted from the figure. The outputelectrodes 33 and 34 are configured such that current flowsperpendicularly to magnetic flux formed roughly perpendicularly to thesurface of the sensor chip and current flowing between the source anddrain electrodes.

The operation of the semiconductor Hall device 2 is described hereafter.Bias is applied to the gate electrode 30, the source electrode 31 andthe drain electrode 32, to set the semiconductor Hall device 2 in thesame operating state as a MOSFET. Preferably, at this time, the deviceoperates in a linear region. When an externally applied magnetic flux isnot present in this state, the two output electrodes 33 and 34 are atthe same potential. When a magnetic flux that is perpendicular to thesurface of the semiconductor Hall device is applied from outside, avoltage that is proportionate to the magnetic flux density arises as adifferential voltage between the output electrodes 33 and 34.

Next, referring to FIG. 5, a method will be described for selectingindividual semiconductor Hall devices that are arranged in an array andextracting the output thereof.

A source electrode, drain electrode and pair of output electrodes ofindividual Hall devices (E(0,0), E(0,1) . . . ) are connected to V_(L),V_(H), OUT 1 and OUT 2 via switches (R0, R1 . . . ), and are connectedcommonly to the same column in the column direction Y. The gateelectrodes of the same row in the row direction X are also common, andare connected to the common gate electrode wire C0, C1 . . . of eachcolumn. V_(L) and V_(H) are wires for supplying bias to the Hall deviceand OUT 1 and OUT 2 are wires for sending output from the Hall devicesto the amplification circuit.

The case of selecting the Hall device E(0,0) will now be described. Onlythe switch R0 is turned ON, and switches R1, R2 . . . are turned OFF.Further, only the gate electrode line C0 is set to a voltage whereby theHall device enters an operating state, and the gate electrode lines C1,C2 . . . are set to a voltage whereby the Hall devices do not operate,i.e. to a state whereby a current does not flow between the source anddrain electrodes even if a bias is applied to the source electrode andthe drain electrode.

At this point, V_(L) and V_(H) are applied to the source electrode anddrain electrode of the Hall device E(0,0) and the Hall devices in thesame row, however current flows only through the Hall device E(0,0). Avoltage that is proportionate to the magnetic flux density arises in theoutput electrodes of the Hall device E (0,0). Because the outputelectrodes of the Hall devices arrayed in the same row as the Halldevice E(0, 0) are not in an operating state, the output voltage of theHall device E(0,0) is output as it is to OUT 1 and OUT 2. With thisconfiguration, even if the number of arrays increases the number ofwires within an array will be the same and it will be only necessary toadd a switch to the end. Thus, the area of the sensor chip will beroughly in proportion to the number of arrays, thereby enabling easyconfiguration of a sensor chip comprising a large number of Halldevices.

FIG. 6 shows the configuration for an entire biosensor. The compositionof the biosensor includes the sensor chip 1 for introducing a samplesolution and carrying out measurement, and a measurement apparatus mainunit that exchanges signals with the sensor chip 1. A semiconductor Halldevice array 9, an array selection circuit 71 and an amplificationcircuit 81 are provided on the sensor chip 1. Other control circuits 82such as, for example, a sensor chip control circuit to control thesensor chip and a signal processing circuit that processes an outputsignal from the Hall devices, are provided on the side of themeasurement apparatus main unit. The sensor chip 1 may be replaced witha new sensor chip after each measurement.

Next, the circuit operation of the entire biosensor according to thepresent invention will be described using the flowchart in FIG. 7 whilereferring to FIG. 6.

In a step S101, in a state where molecular receptors, objects to bemeasured, and magnetic molecules including magnetic particles have beenintroduced onto a sensor chip, a magnetic field is applied from a lowercoil. The magnetic molecules are attracted to the surface of the sensorchip by the magnetic field that is produced such that the magnetic fluxdensity decreases as the distance from the surface of the sensor chipincreases, thereby enhancing the speed of binding to the surface of thesensor chip.

In a step S102, in a state where binding of magnetic molecules to thesurface of the sensor chip is completed, the magnetic field originatingfrom the lower coil is turned OFF.

In a step S103, in a state where a magnetic field is not applied to thesensor chip, the output of Hall devices is obtained. Specifically, anaddress signal for selecting a specific Hall device is sent from asensor chip control circuit 82 in the measurement apparatus main unit tothe array selection circuit 71 on the sensor chip. Based on this addresssignal the array selection circuit 71 selects the specified Hall deviceas described above. An output signal from that Hall device is amplifiedby the amplification circuit 81 on the sensor chip, and stored in amemory 83 as an offset value (first output value).

In a step S104, the sensor chip control circuit judges whether signalshave been acquired from all the Hall devices, from which output signalsare required, and if all the signals have not been acquired it returnsto the step S103. Thus, the output signals of all of the Hall devicesare extracted and recorded.

In a step S105, a magnetic field is applied from an upper coil.

In a step S106, as described above, address information of a Hall deviceis sent to the sensor chip from the sensor chip control circuit 82, anoutput signal is extracted, and similarly to the step S103, the signalis stored as the output value of the Hall device (second output value)in the memory 83.

In a step S107, as described above, the sensor chip control circuitjudges whether signals have been acquired from all the Hall devices,from which output signals are required, and if all the signals have notbeen acquired it returns to the step S106. Thus, the binding state ofmagnetic molecules with respect to all the Hall devices is acquired.

In a step S108, the magnetic field of the upper coil is turned OFF.

In a step S109, the offset values of the respective Hall devicesacquired in the step S103 and the corresponding output values of theHall devices acquired in the step S106 are retrieved from the memory 83,and the output values of the Hall devices are corrected using the offsetvalues in the signal processing circuit 82.

In a step S110, the output values after correction in the step S109 arecompared with respect to adjacent Hall devices. If the states ofadjacent Hall devices are the same, that is, both have magneticmolecules bound thereto or neither has a magnetic molecule boundthereto, the output values will be the same. When the states of adjacentHall devices are different, that is, only one of the Hall devices has amagnetic molecule bound thereto, the output value of the Hall devicehaving a magnetic molecule bound thereto will be larger than that of theHall device not having a magnetic molecule bound thereto. This isbecause the magnetic flux is concentrated by the magnetic molecule.

By comparing the output value of each of the Hall devices with respectto the output value of the Hall device adjacent thereto, the boundariesbetween regions in which magnetic molecules are bound and regions inwhich magnetic molecules are not bound can be determined. Thus, it ispossible to determine the number of magnetic molecules bound on thesensor chip surface. The above comparison is not limited to comparisonbetween output values of adjacent semiconductor Hall devices, and anycomparison may be performed as long as the output values used forcomparison are those obtained from semiconductor Hall devices providedin mutually different regions.

Further, a specified Hall device that does not comprise on its surfacemolecular receptors that capture objects to be measured may be providedas a reference region to which magnetic molecules do not bind.

In FIG. 7, after acquiring the offset values of all the Hall devices,the upper coil is turned ON and the output values of all the Halldevices are then acquired, however it is also possible to acquire theoffset value and then acquire the output value for each Hall device,respectively. That is, after executing the step S103 for one Halldevice, the steps S105, S106 and S108 are then executed in that order,and after repeating this procedure for each of the Hall devices, thesteps S109 and S110 are executed.

The biosensor is controlled using a program for implementing theoperations of FIG. 7 as described above. More specifically, the programis a program for controlling a biosensor which includes a measurementstep for acquiring the intensities of magnetic fields appliedrespectively to different regions on the surface of a magnetic sensor,and a determination step for determining the amount of bound magneticmolecules by comparing the intensities of the magnetic fields appliedrespectively to the mutually different regions that were acquired in themeasurement step.

In the measurement step, each of the individual detector elementsarranged in a two-dimensional array is selected, and the intensity ofthe magnetic field detected by each individual detector element isacquired.

It is preferable that the program further comprises an offset valueacquisition step for acquiring the outputs from the above detectorelements as offset values before a magnetic field is applied to thesurface of the magnetic sensor in the above measurement step, and thatin the above determination step, comparison is conducted using a valueobtained by removing the offset values acquired in the offset valueacquisition step from the values acquired from the detector elements inthe measurement step.

Also, it is preferable that the program further comprises a bindingacceleration step for generating, upon introduction of the magneticmolecules to the surface of the magnetic sensor, a magnetic field forbringing magnetic molecules close to the surface of the magnetic sensorby a magnetic field generation means provided below the surface of themagnetic sensor to which the magnetic molecules bind, and that the speedof binding of the magnetic molecules in the binding acceleration step isenhanced by a magnetic field produced such that the magnetic fluxdensity decreases according to an increase in the distance from thesurface of the magnetic sensor.

The biosensor control program can be used by recording the program inthe aforementioned memory provided in the measurement apparatus mainunit of the biosensor or in a read-only storage device provided in themeasurement apparatus main unit, or by storing the program in a storagedevice of another computer or the like.

Embodiment 2

Next, the circuit operation of the entire biosensor according to thesecond embodiment of the present invention will be described using theflowchart shown in FIG. 8. The configuration of the entire biosensor isthe same as that shown in FIG. 6. However, the amplification circuit 81in FIG. 6 further comprises a detector circuit for extracting only afrequency component from the output signals of the semiconductor Halldevices.

In a step S201, in a state where objects to be measured and magneticmolecules that include magnetic particles have been introduced onto asensor chip, a magnetic field is generated by passing a direct currentthrough a lower coil, whereby the magnetic molecules are attracted tothe sensor chip surface.

In a step S202, the magnetic field originating from the lower coil isturned OFF.

In a step S203, a magnetic field is generated by passing a directcurrent through an upper coil, whereby magnetic molecules are attractedaway from the sensor chip surface.

In a step S204, the magnetic field originating from the upper coil isturned OFF.

In a step S205, the operation returns to the step S201 and the stepsfrom S201 to S204 are repeated until the lapse of a pre-established timefor completion of binding of magnetic molecules to the sensor chipsurface or the completion of a pre-established number of steprepetitions.

In a step S206, an alternating current magnetic field is generated bypassing an alternating current through the upper coil.

In a step S207, the output signal of each Hall device is acquired.Specifically, an address signal for selecting a specific Hall device issent from the sensor chip control circuit 82 that is provided on themeasurement apparatus main unit side to the array selection circuit 71provided on the sensor chip. Based on this address signal the arrayselection circuit 71 selects the specified Hall device as describedabove. The amplification circuit 81 on the sensor chip amplifies theoutput signal from the Hall device. As described above, theamplification circuit 81 comprises a detector circuit for extractingonly a frequency component corresponding to the frequency of the appliedmagnetic field from the output signal. The output signal extracted bythe detector circuit is stored in the memory 83 after amplification.

In a step S208, as described above, judgment is made as to whethersignals have been acquired from all of the Hall devices, from whichoutput signals should be acquired. If a signal has not been acquiredfrom all of the Hall devices the operation returns to the step S207.Thus, the output signal of each Hall device is acquired.

In a step S209, the magnetic field of the upper coil is turned OFF.

In a step S210, the output value of each Hall device that was acquiredin the step S207 is retrieved from the memory 83 and compared with theoutput value of the Hall device adjacent thereto in the signalprocessing circuit 82. If the states of adjacent Hall devices are thesame, that is, both have magnetic molecules bound thereto or neither hasa magnetic molecule bound thereto, the output values will be the same.When the states of adjacent Hall devices are different, that is, onlyone of the Hall devices has a magnetic molecule bound thereto, theoutput value of the Hall device having a magnetic molecule bound theretowill be larger than the output value of the Hall device not having amagnetic molecule bound thereto. This is because the magnetic flux isconcentrated by the magnetic molecule, thus causing an increase in theoutput value of the Hall device having a magnetic molecule boundthereto.

By comparing the output value of each Hall device with the output valueof the Hall device adjacent thereto, the boundaries between regions inwhich magnetic molecules are bound and regions in which magneticmolecules are not bound can be determined. Thus, it is possible todetermine the number of magnetic molecules bound on the sensor chipsurface.

The biosensor is controlled using a program for implementing theoperations of FIG. 8 as described above. More specifically, the programis a program for controlling the aforementioned biosensor in which, inthe above offset value correction step, using a magnetic field appliedat a constant frequency across the above magnetic sensor, by retrievingonly a frequency component corresponding to the magnetic field from anoutput signal of a detector element that includes a signal output at afrequency corresponding to the magnetic field, an offset signal includedas a direct current component can be removed from the output signal.

That is, the output signal of the detector element comprises analternating current signal that corresponds to the frequency of anapplied alternating current magnetic field and a direct current offsetsignal that is output regardless of the application of a magnetic field.By means of the detector circuit, an offset value included as a directcurrent component in the output signal of a detector element can beeliminated by acquiring only a frequency component for an appliedalternating current magnetic field from the output signal of thedetector element.

It is preferable that the program further comprises a stirring step tobe conducted after the introduction of the magnetic molecules to thesurface of the magnetic sensor and prior to the above measurement step.In the stirring step, magnetic molecules are stirred by alternatelyproducing magnetic fields by means of a first magnetic field generationmeans provided in a position facing the surface of the magnetic sensorand a second magnetic field generation means provided below the surfaceof the magnetic sensor.

Preferably, in the above stirring step, by alternately inverting theinclination of the magnetic flux density formed in a perpendiculardirection to the surface of the magnetic sensor, the speed of bindingbetween an object to be measured and a magnetic molecule is enhanced,and the speed of binding between a molecular receptor immobilized on thesurface of the magnetic sensor and a magnetic molecule bound with anobject to be measured is also enhanced.

With respect to the description of the scope of the claims, the presentinvention may take the following form.

(1) The biosensor according to claim 13, which further comprises amagnetic field switching means for performing switching such that thefirst magnetic field generation means and the second magnetic fieldgeneration means alternately generate a magnetic field, characterized inthat magnetic molecules are stirred by alternately changing a state ofdistribution of a magnetic field on a magnetic sensor surface by meansof the magnetic field switching means.

Thereby, magnetic particles can be stirred in a sample solution, and thespeed of binding between magnetic particles used as labels inmeasurement and other substances can be enhanced, and thus a timerequired for measurement can be reduced.

As used herein, a magnetic field switching means may be composed of, forexample, a switch for alternately passing current to a first coil usedas a first magnetic field generation means and a second coil used as asecond magnetic field generation means. The switching frequency andswitching time and the like may be set up in advance.

Embodiment 3

Next, the detection principles of a biosensor according to the thirdembodiment of the present invention will be described using FIG. 9. FIG.9 is a view schematically showing a cross section of the vicinity of thesemiconductor Hall device 2 of the sensor chip 1. The molecularreceptors 61 composed of antibodies are immobilized on the surface ofthe semiconductor Hall device 2. The object to be measured 62 is boundspecifically to one of the molecular receptors 61. Further, the magneticparticle 51 is bound to the object to be measured 62 through specificbinding between the object to be measured 62 and one of the molecularreceptors 63 that are composed of antibodies. The magnetic particle 51and the molecular receptors 63 bind to each other to form the magneticmolecule 5.

The sensor chip 1 is arranged with its surface facing downward, and arear coil CR (second magnetic field generation means) is provided. Theintensity of a magnetic field generated by the rear coil CR is set suchthat the intensity is sufficient for attracting magnetic molecules tothe surface of the sensor chip 1. As shown in FIG. 9, part of a magneticmolecules binds to the surface of the sensor chip 1. In this state, themagnetic field generated by the rear coil CR is weakened to a degreesuch that floating magnetic molecules that are not bound to the surfaceof the sensor chip 1 sink downward due to gravitational force.

In FIG. 9, the magnetic flux B is formed in the direction shown by anarrow Z2, which is perpendicular with respect to the surface of thesemiconductor Hall device. Since the magnetic flux B is concentrated atthe magnetic particle 51, the magnetic flux density at the semiconductorHall device 2 is increased in comparison to a case where the magneticparticle 51 is not present. At this point, magnetic molecules 5 thathave sunken downward do not affect the magnetic flux density detected bythe semiconductor Hall device 2. Since the output voltage of thesemiconductor Hall device 2 is in proportion to the magnetic fluxdensity, whether or not a magnetic molecule 5 is bound on thesemiconductor Hall device 2 can be determined based on the outputvoltage.

In this embodiment it is preferable that at the time of introduction ofmagnetic molecules to the sensor chip 1, the rear coil CR intermittentlygenerates a magnetic field of an intensity sufficient for attractingmagnetic molecules to the surface of the sensor chip 1, such that thespeed of binding between objects to be measured and magnetic particlesis enhanced by stirring in which the magnetic particles are moved up anddown, and further, the speed of binding of magnetic particles bound withobjects to be measured to molecular receptors that are immobilized onthe magnetic sensor surface is also enhanced.

The state of a magnetic field applied to magnetic molecules by the coilaccording to Embodiment 3 is shown in FIG. 10.

First, magnetic molecules are stirred to facilitate reaction betweenmolecular receptors and objects to be measured. During the period fromstirring start T₁ to stirring end T₂, the rear coil CR intermittentlyapplies a magnetic field having a magnetic field intensity B₁ that isstrong enough to attract magnetic molecules. In the figure, thegenerated magnetic field is bipolar, however the magnetic field may beunipolar. After completion of reaction, in a measurement step (frommeasurement start T₃ to measurement end T₄) the rear coil CR generates aweak alternating current magnetic field of a magnetic field intensity B₂that is of an intensity such that the magnetization of magneticmolecules does not become saturated, and the signals of all magneticsensors are measured.

Next, the circuit operation of the entire biosensor according toEmbodiment 3 of the present invention will be explained using theflowchart in FIG. 11. The configuration of the entire biosensor is thesame as that shown in FIG. 6, except that the amplification circuit 81in FIG. 6 further comprises a detector circuit for extracting only afrequency component from the output signal of a semiconductor Halldevice.

In a step S301, in a state where objects to be measured and magneticmolecules that include magnetic particles have been introduced to asensor chip, a magnetic field is generated bypassing intermittentcurrent through the rear coil, whereby magnetic molecules are attractedto the sensor chip surface during periods when the current is flowing.

In a step S302, the operation returns to the step S301 and the stepsS301 and S302 are repeated until a pre-established time for completionof binding of magnetic molecules to the sensor chip surface has lapsed.

In a step S303, the magnetic field originating from the rear coil isturned OFF.

In a step S304, an alternating current magnetic field is applied bypassing alternating current through the rear coil.

In a step S305, the output signal of each Hall device is acquired.Specifically, an address signal for selecting a specific Hall device issent from the sensor chip control circuit 82 that is provided on themeasurement apparatus main unit side to the array selection circuit 71provided on the sensor chip. Based on this address signal, the arrayselection circuit 71 selects the specified Hall device as describedabove. The output signal from the Hall device in question is amplifiedby the amplification circuit 81 on the sensor chip. As described above,the amplification circuit 81 comprises a detector circuit for extractingonly a frequency component corresponding to the frequency of the appliedmagnetic field from the output signal. The output signal extracted bythe detector circuit is stored in the memory 83 after amplification.

In a step S306, as described above, judgment is made as to whethersignals have been acquired from all of the Hall devices, from whichoutput signals should be acquired. If a signal has not been acquiredfrom each of the Hall devices the operation returns to the step S305.Thus, the output signals of all the Hall devices are acquired.

In a step S307, the magnetic field of the rear coil is turned OFF.

In a step S308, the output value for each Hall device that was acquiredin the step S305 is retrieved from the memory 83. Then, in the signalprocessing circuit 82, comparison is carried out between output valuesof Hall devices that do not comprise molecular receptors on theirsurface, that is, Hall devices arranged in a reference region, andoutput values of Hall devices that comprise molecular receptors on theirsurface. If a Hall device that comprises a molecular receptor on itssurface does not have a magnetic molecule binding thereto, the outputvalue thereof will be at the same level as the output value of a Halldevice arranged in the reference region. If a Hall device has a magneticmolecule binding thereto, the output value thereof will be larger thanthat of a Hall device in the reference region since the magnetic fluxwill be concentrated by the magnetic molecule.

By comparing the respective output values of all the Hall devices havingmolecular receptors on their surface with the output value of a Halldevice that does not have a molecular receptor on its surface, thenumber of magnetic molecules bound on the sensor chip can be determined.

FIG. 12 is a view showing the state of disposition of Hall devices inthis embodiment.

In the figure, of Hall devices 2 a, 2 b, and 2 c, only the Hall devices2 a and 2 c comprise molecular receptors on their surface.

When the size of the Hall voltage, which is the voltage differencebetween two output terminals of the Hall device, is represented by “VD”,and the size of the magnetic flux density of a magnetic field applied tothe Hall device is represented by “B”, the following formula (1)applies:VD=A×B  Formula (1) (A is a proportionality factor)

When the size of the magnetic flux density of a magnetic field appliedto a Hall device by a rear coil in a state where magnetic molecules arenot bound to the surface thereof is taken as “B0”, and the size of themagnetic flux density in a state where magnetic molecules are bound tothe surface thereof is taken as “B0 (1+Δ)”, the sizes VD_(2a), VD_(2b),VD_(2c), of the Hall voltage of the respective Hall devices 2 a, 2 b and2 c are respectively represented in the following formulas (2) to (4):VD _(2a) =A×B 0(1+Δ)  Formula (2)VD _(2b) =A×B 0  Formula (3)VD _(2c) =A×B 0  Formula (4)When the sensitivity of the Hall device, i.e. “A”, is unchanging and thesize “B0” of the magnetic flux density of a magnetic field applied tothe Hall device by a coil is also constant, the presence or absence ofbinding of a magnetic molecule on the surface of each Hall device can bedetermined from the absolute value of the Hall voltage. However,although in theory the sensitivity and the size of the magnetic fluxdensity applied by a coil will be the same for adjacent Hall devices,because of variations in the production process or variations in thedistance between the coil and the Hall devices and the like the absolutevalue thereof is not always constant. Therefore, for individual Halldevices it is difficult to determine the presence or absence of bindingof a magnetic molecule on the surface thereof using only the Hallvoltage thereof.

If the size of the Hall voltage of the Hall device 2 b is taken as areference, and the difference thereof with the Hall voltages of the Halldevices 2 a and 2 b is calculated, the resulting values will be thoseshown respectively in formulas (5) and (6).VD _(2a) −VD _(2b) =A×B 0×Δ  Formula (5)VD _(2c) −VD _(2b)=0  Formula (6)Thus, by employing as a reference the Hall voltage of a Hall device in areference region, which is a Hall device not comprising a molecularreceptor on the surface thereof, even if the absolute value of themagnetic flux density or the sensitivity fluctuates, the presence orabsence of binding of a magnetic molecule can be identified based onwhether or not the difference is zero.

The difference can also be determined in this way in the comparison todetermine the presence or absence of binding. For example, as describedhereafter, the presence or absence of binding can also be determinedbased on a value obtained by dividing the Hall voltage that is theobject for comparison by the Hall voltage of a reference region.

As described in the foregoing, a reference region may be provided, forexample, by previously providing an unformed region of a gold thin filmon a sensor surface. Thereby, at the time of immobilizing molecularreceptors, molecular receptors are not immobilized on the referenceregion, and thus a region to which the magnetic particles 51 areincapable of binding is formed. The number of reference regions on asensor surface is not limited to one, and a plurality of referenceregions may be provided thereon.

The biosensor is controlled using a program for implementing theoperations in FIG. 11 as described above.

More specifically, the program is a program for controlling theaforementioned biosensor in which, in the above offset value correctionstep, using a magnetic field applied at a constant frequency across theabove magnetic sensor, by retrieving only a frequency componentcorresponding to the magnetic field from an output signal of a detectorelement that includes a signal output at a frequency corresponding tothe magnetic field, an offset signal included as a direct currentcomponent can be removed from the output signal.

That is, the output signal of the detector element comprises analternating current signal that corresponds to the frequency of anapplied alternating current magnetic field and a direct current offsetsignal that is output regardless of the application of a magnetic field.Using the detector circuit, an offset value included as a direct currentcomponent in the output signal of a detector element can be eliminatedby acquiring only the component of the frequency of the appliedalternating current magnetic field from the output signal of thedetector element.

Further, at the time of introduction of the magnetic molecules to thesurface of the magnetic sensor, a magnetic field generation means thatgenerates a magnetic field for drawing the magnetic molecules close tothe magnetic sensor surface is intermittently activated to enablestirring of the magnetic molecules.

In the aforementioned stirring step, by producing a magnetic fluxintermittently in a direction perpendicular to the surface of themagnetic sensor, the speed of binding between objects to be measured andmagnetic molecules can be enhanced. Further, the speed at which amagnetic molecule bound with an object to be measured comprising anantigen binds through the object to be measured with a molecularreceptor immobilized on the magnetic sensor surface that captures theobject to be measured can be enhanced.

FIG. 13 is a table showing values obtained by dividing the output valuesof five Hall devices having on their surfaces molecular receptorscapturing objects to be measured by the output value of a Hall devicenot having a molecular receptor on its surface, in which the respectiveoutput values were obtained in a measurement step.

In this measurement, Hall devices having the form shown in FIGS. 4A to4C were arranged in an array, and an array selection circuit andamplification circuit were fabricated on the same silicon substrate. Thedistance between the source electrode 31 and drain electrode 32 of theHall device was approximately 6.4 μm, and the distance from the sensingsurface, that is, from a channel formed below the gate electrode 30 tothe surface of the insulating layer 12, was approximately 2.8 μm. Thedisposition pitch of the Hall devices arranged in the array was 12.8 μm.A voltage between the source electrode and drain electrode of a Halldevice selected by the array selection circuit was approximately 4 V,and a voltage between the source electrode and gate electrode wasapproximately 5 V. The magnetic flux density generated on the surface ofthe Hall devices by a coil was 20 Hz, and approximately 50 grms.Magnetic particles manufactured by Dynal Biotech (product name:Dynabeads) having a diameter of 4.5 μm were used as the magneticparticles.

Referring to FIG. 13, the output of semiconductor Hall devices No. 2,No. 4, and No. 5 is approximately 5% larger than the output of asemiconductor Hall device not having a molecular receptor on itssurface. Therefore, it could be assumed that magnetic particles werebound to the semiconductor Hall devices No. 2, No. 4, and No. 5. Inverification using a microscope it was found that magnetic particleswere bound to the surfaces of semiconductor Hall devices No. 2, No. 4,and No. 5, and magnetic particles were not bound to the surfaces ofsemiconductor Hall devices No. 1 and No. 3. This matched the resultsshown in FIG. 13.

Embodiment 4

FIG. 14 schematically shows a cross section of a sensor chip used inEmbodiment 4.

In FIG. 14, a magnet is arranged above the sensor chip 1. At this point,the magnetic flux B originating from the magnet is formed in thedirection shown by the arrow Z that is perpendicular to the surface ofthe semiconductor Hall device. Because the magnetic flux B isconcentrated at the magnetic particle 51, the magnetic flux density atthe semiconductor Hall device 2 is increased in comparison to a casewhere the magnetic particle 51 is not present. Since the output voltageof the semiconductor Hall device 2 is in proportion to the magnetic fluxdensity, it is possible to determine whether or not the magneticmolecule 5 is bound on the semiconductor Hall device 2 based on theoutput voltage.

Using the flowchart shown in FIG. 15, the circuit operation of theentire biosensor according to Embodiment 4 that uses the biosensor shownin FIG. 14 will be described. The configuration of the entire biosensorused herein is the same as that shown in FIG. 6.

In a step S401, in a state where objects to be measured and magneticmolecules that include magnetic particles are not introduced onto asensor chip, a magnetic field is applied by a magnet and the intensityof a magnetic field prior to binding of magnetic molecules is acquired.Specifically, an address signal for selecting a specific Hall device issent from a sensor chip control circuit 82 that is located on themeasurement apparatus main unit side to an array selection circuit 71 onthe sensor chip. Based on this address signal, the array selectioncircuit 71 selects the specified Hall device as described above. Theoutput signal from the Hall device in question is amplified by theamplification circuit 81 on the sensor chip, and stored in the memory 83as an initial value.

In a step S402, judgment is made as to whether signals have beenacquired from all the Hall devices, from which output signals should beacquired. If a signal has not been acquired from each Hall device theoperation returns to the step S401. Thus, the output signal of each Halldevice is extracted and recorded.

In a step S403, objects to be measured and magnetic molecules areintroduced onto a sensor chip in a state where a magnetic field is notapplied thereto, and upon completion of binding a magnetic field of thesame intensity as that in the step S401 is applied by the magnet. Inthis state, as described above, address information of a Hall device issent from the sensor chip control circuit 82 to the sensor chip, and theoutput signal of the relevant Hall device is extracted.

In a step S404, the initial value of the same Hall device that wasacquired in the step S401 is retrieved from the memory 83. In the signalprocessing circuit 82, the output signal extracted in the step S403 andthe initial value are compared.

In a step S405, the result of the comparison conducted in the step S404is output. Since the output of a Hall device to which a magneticmolecule is bound will be different to the initial value thereof,irrespective of the fact that magnetic fields of the same intensity wereapplied thereto, it is thus possible to determine whether or not amagnetic molecule is bound on a Hall device in an arbitrary position.Also, as described hereafter, by obtaining a comparison result for eachof the Hall devices, the number of magnetic molecules bound on thesensor chip can be determined. The comparison results can be output asthe number of magnetic molecules bound on a sensor chip or as positioninformation, according to the purpose of use.

In a step S406, as described above, judgment is made as to whethersignals have been acquired from all the Hall devices, from which outputsignals should be acquired. If a signal has not been acquired from eachHall device the operation returns to the step S403. Thus, the state ofbinding of magnetic molecules on each of the Hall devices is acquired.

The biosensor is controlled using a program for implementing theoperations in FIG. 15 as described above. More specifically, the programis a program for controlling the aforementioned biosensor that includes:a pre-binding measurement step for acquiring the intensity of a magneticfield applied to a magnetic sensor prior to binding of magneticmolecules; a post-binding measurement step for acquiring the intensityof a magnetic field applied to a magnetic sensor after binding ofmagnetic molecules; and a determination step for determining the amountof bound magnetic molecules by comparing the intensity of the magneticfield prior to binding with the intensity of the magnetic field afterbinding. Further, in the pre-binding measurement step and thepost-binding measurement step, each of the detector elements arranged ina two-dimensional array is selected, and the intensity of the magneticfield detected by each detector element is acquired. The biosensorcontrol program can be used by recording the program in theaforementioned memory provided in the measurement apparatus main unit ofthe biosensor or in a read-only storage device provided in themeasurement apparatus main unit, or by storing the program in a storagedevice of another computer or the like.

FIG. 16 shows the values for output signals prior to binding and outputsignals after binding.

In this measurement, Hall devices having the form shown in FIGS. 3A to3C were arranged in an array and an array selection circuit andamplification circuit were fabricated on the same silicon substrate. Thedistance between the source electrode 31 and drain electrode 32 of theHall devices was approximately 6.4 μm, and the distance from the sensingsurface, that is, from a channel formed below the gate electrode 30 tothe surface of the insulating layer 12 was approximately 5 μm. Thedisposition pitch of the Hall devices arranged in the array was 12.8 μm.The gain of the amplification circuit was 100-fold. A voltage betweenthe source electrode and the drain electrode of a Hall device selectedby the array selection circuit was approximately 4V, and a voltagebetween the source electrode and the gate electrode was approximately 5V. The magnet used was such that a magnetic flux density ofapproximately 2500 gauss was generated across the surface of the Halldevice. The magnetic particles used were manufactured by Dynal Biotech(product name: Dynabeads) and had a diameter of 4.5 μm.

FIG. 16 shows the values of output signals from five Hall devicesarranged in an array, before and after binding of magnetic molecules.Referring to FIG. 16, for semiconductor Hall devices No. 1 to No. 5arranged in an array at respectively different positions, a pre-bindingoutput voltage (mV), a post-binding output voltage (mV) and a voltagedifference (mV) between the two are, respectively, (882, 882, 0) forsemiconductor Hall device No. 1; (886, 887, 1) for semiconductor Halldevice No. 2; (885, 885, 0) for semiconductor Hall device No. 3; (887,892, 5) for semiconductor Hall device No. 4; and (886, 887, 1) forsemiconductor Hall device No. 5. As is clear from the differencesbetween the values for pre-binding and post-binding, the signal fromsemiconductor Hall device No. 4 to which magnetic molecules attached wasthe only signal that increased. Therefore it could be assumed thatmagnetic molecules bound only to the semiconductor Hall device No. 4. Inconfirmation using a microscope prior to binding and after binding onthe Hall devices, it was found that magnetic molecules bound only tosemiconductor Hall device No. 4, and magnetic molecules did not bind toany of the other semiconductor Hall devices. This matched the resultsfor the output signals.

The above description relates to specific embodiments of the presentinvention. A person skilled in the art can suitably devise variousmodifications of the present invention, and such modifications are alsoencompassed in the technical scope of the present invention.

Industrial Applicability

By using the biosensor according to the present invention, it ispossible to determine the two-dimensional distribution of objects to bemeasured or the amount of bound objects to be measured.

In particular, by determining the amount of an object to be measured bycomparing the intensities of magnetic fields of respectively differentregions on the surface of a magnetic sensor, measurement can beperformed promptly. This is because the value of a magnetic field to bethe object for comparison and the value of a magnetic field to be thereference for comparison can be acquired in the same state, that is, ina state where magnetic molecules and objects to be measured have beenintroduced onto the magnetic sensor. Further, by obtaining an outputvalue in a reference region to which magnetic molecules cannot bind andcomparing that value with the output value of each detector element,precise assessment of the presence or absence of binding is enabledirrespective of fluctuations in the sensitivity of the sensor orfluctuations in the absolute value of the magnetic flux density or thelike, and detection of minute changes in a magnetic field is alsoenabled. In addition, with respect to offset values that depend on theconfiguration of a sensor, values can be acquired having conditions of asample solution in which magnetic molecules or the like are introducedand operating conditions of an apparatus and the like equivalent tothose at the time of measurement, whereby measurement of a higheraccuracy is possible.

By making the size of the surface of semiconductor Hall devicesequivalent to or less than the size of magnetic molecules and making theintervals at which the semiconductor Hall devices are arranged largerthan the size of the magnetic particles, interference from othermagnetic molecules can be eliminated. This can enhance the accuracy ofdetection and analysis.

Further, by selectively immobilizing molecular receptors on the surfaceof a magnetic sensor, for example, by immobilizing molecular receptorson sections corresponding to the sites of semiconductor Hall devices,the detection state of magnetic molecules binding to molecular receptorscan be adjusted.

Forming a magnetic sensor using semiconductor Hall devices according tothe present invention makes it possible to use common interconnections,and thus the number of devices can be easily increased and constructionof a low-cost, small-size biosensor is enabled.

Further, by attracting magnetic molecules that are not bound to a sensorsurface away from the sensor surface utilizing a magnetic field, it ispossible to carry out fast and accurate measurement without the need towash away floating magnetic molecules that have not bound to themagnetic sensor surface when determining the quantity of bound magneticmolecules.

While measurement may be conducted after removing unbound magneticmolecules using a magnet or the like, constructing a biosensor in whichthe sensor surface is facing downward allows measurement to be performedat the same time as isolation of unbound molecules. In addition, it alsoallows the intensity of a magnetic field to be applied at the time ofmeasurement to be kept to a level whereby the magnetic field does notbecome saturated, thereby enabling output signals to be obtained fromHall devices to be more obvious.

By providing a magnetic field generation means that attracts magneticmolecules to the surface of the magnetic sensor, it is possible to speedup binding of magnetic molecules to the magnetic sensor surface, therebyreducing the assay time.

Further, by forming a magnetic sensor, selection means, and signalamplification circuit on one chip, it is possible to miniaturize themagnetic sensor and, at the same time, to replace one magnetic sensorwith another according to a sample solution used.

1. A biosensor which performs analysis of an object to be measured bymeans of a magnetic sensor composed of detector elements for detecting amagnetic field produced by bound magnetic molecules, the detectorelements being arranged two-dimensionally in X rows and Y columns (X andY are natural numbers, the same applies hereunder), and the analysis ofan object to be measured being conducted by measuring an amount of themagnetic molecules, characterized in that the detector element comprisesa semiconductor Hall device and the biosensor comprises a signalprocessing means which determines an amount of the bound magneticmolecules by comparing intensities of magnetic fields of differentregions on a sensor surface of the magnetic sensor, and analysis of anobject to be measured is conducted based on the determined amount ofmagnetic molecules.
 2. The biosensor according to claim 1, characterizedin that a reference region to which the magnetic molecules are incapableof binding is provided on the sensor surface of the magnetic sensor, andthe signal processing means conducts comparison using an intensity of amagnetic field of the reference region as a reference.
 3. The biosensoraccording to claim 2, characterized in that surface treatment isperformed for the reference region such that molecular receptors thatbind with the magnetic molecules are incapable of immobilizing thereto.4. The biosensor according to any one of claims 1 to 3, characterized inthat the biosensor further comprises a selection means for selectingindividual detector elements arranged in the X rows and Y columns andextracting an output thereof.
 5. The biosensor according to claim 4,characterized in that the magnetic sensor, the selection means, and asignal amplifier circuit that amplifies an output signal of the detectorelement are formed on one chip.
 6. The biosensor according to any ofclaim 1, characterized in that the size of each detection space in whichdetection of a magnetic field by the detector element is possible isequal to the size of approximately one molecule of the magneticmolecules for binding.
 7. The biosensor according to claim 1,characterized in that in the magnetic sensor the detector elements arearranged at intervals such that two detector elements next to each otherdetect mutually different magnetic molecules.
 8. The biosensor accordingto claim 7, characterized in that the detector elements which areadjacent are arranged adjacently at an interval that is equal to orgreater than the diameter of the magnetic molecules.
 9. The biosensoraccording to claim 1, characterized in that the sensor surface of themagnetic sensor has been subjected to surface treatment for selectivelyimmobilizing in specified regions molecular receptors that bind with themagnetic molecules.
 10. The biosensor according to claim 9,characterized in that recesses of a size corresponding to a size of themagnetic molecules are provided on the sensor surface of the magneticsensor, and molecular receptors that bind to the magnetic molecules areprovided only in the recesses on the sensor surface of the magneticsensor.
 11. The biosensor according to claim 9 or 10, characterized inthat a gold thin film is formed in the specified regions on the sensorsurface of the magnetic sensor, and the molecular receptors that have anend modified by a thiol group are selectively immobilized thereon. 12.The biosensor according to claim 1, characterized in that the biosensorcomprises a first magnetic field generation means arranged in a positionfacing the sensor surface of the magnetic sensor that generates amagnetic field that is applied to the sensor surface of the magneticsensor.
 13. The biosensor according to claim 12, characterized in thatthe biosensor comprises means whereby the first magnetic fieldgeneration means comprises means for intermittently generating amagnetic field.
 14. The biosensor according to claim 12 or 13,characterized in that the first magnetic field generation meanscomprises a detector circuit for generating a magnetic field at aconstant frequency, and extracting only a frequency componentcorresponding to the magnetic field from an output signal of thedetector element.
 15. The biosensor according to claim 1, characterizedin that the biosensor comprises a second magnetic field generation meansarranged on the backside of the sensor surface of the magnetic sensorthat generates a magnetic field that is applied to the sensor surface ofthe magnetic sensor.
 16. The biosensor according to claim 15,characterized in that the biosensor comprises means whereby the secondmagnetic field generation means comprises means for intermittentlygenerating a magnetic field.
 17. The biosensor according to claim 15 orclaim 16, characterized in that the biosensor comprises a detectorcircuit which, when the second magnetic field generation means generatesa magnetic field at a constant frequency, extracts only a frequencycomponent corresponding to the magnetic field from an output signal ofthe detector element.
 18. The biosensor according to any of claim 1,characterized in that the sensor surface of the magnetic sensor isarranged so that the sensor surface of the magnetic sensor is facedtowards a direction in which gravitational force acts.
 19. The biosensoraccording to claim 1, characterized in that the semiconductor Halldevice has a pair of current terminals, a gate electrode controllingcurrent flowing between the current terminals, and a pair of outputterminals arranged such that current flows roughly perpendicular tocurrent flowing between the current terminals.
 20. The biosensoraccording to claim 4, characterized in that the semiconductor Halldevice has a pair of current terminals, a gate electrode for controllingcurrent flowing between the current terminals, and a pair of outputterminals arranged such that current flows roughly perpendicular tocurrent flowing between the current terminals, wherein the gateelectrode is connected to a gate electrode wire that is common to thesemiconductor Hall devices arranged in the same column, the pair ofcurrent terminals are connected to a pair of current terminal wires thatare common to the semiconductor Hall devices arranged in the same row,the pair of output terminals are connected to a pair of output terminalwires that are common to the semiconductor Hall devices arranged in thesame row, and the selection means extracts an output signal of thesemiconductor Hall device arranged in an arbitrary position by selectingthe gate electrode wire, pair of current terminal wires, and pair ofoutput terminal wires.
 21. A method for measuring magnetic molecules inwhich a magnetic sensor composed of detector elements for detecting amagnetic field produced by bound magnetic molecules, in which thedetector elements are arranged two-dimensionally in X rows and Y columns(X and Y are natural numbers, the same applies hereunder), is used tomeasure an amount of the magnetic molecules, characterized in that themethod comprises: a measurement step for acquiring intensities ofmagnetic fields of mutually different regions of a sensor surface of themagnetic sensor; and a determination step for determining an amount ofthe bound magnetic molecules by comparing the intensities of themagnetic fields of the mutually different regions obtained using theintensity of magnetic field of the reference region as a reference inthe measurement step.
 22. A method for measuring magnetic molecules inwhich a magnetic sensor composed of detector elements for detecting amagnetic field produced by bound magnetic molecules, the detectorelements being arranged two-dimensionally in X rows and Y columns (X andY are natural numbers, the same applies hereunder), is used to measurean amount of the magnetic molecules, characterized in that the methodcomprises: a pre-binding measurement step for acquiring intensities ofmagnetic fields prior to binding of the magnetic molecules. apost-binding measurement step for acquiring intensities of magneticfield after binding of the magnetic molecules; and a determination stepfor determining an amount of the bound magnetic molecules by comparingthe intensities of the magnetic fields prior to binding with theintensities of the magnetic fields after binding.
 23. The method formeasuring magnetic molecules according to claim 21 or claim 22,characterized in that the method further comprises an offset valueacquisition step for acquiring an offset value that is output from thedetector element.
 24. The method for measuring magnetic moleculesaccording to claim 21, characterized in that in the measurement step, bymeans of a magnetic field applied at a constant frequency to the sensorsurface, an output signal of a detector element is obtained thatincludes a signal output at a frequency corresponding to the magneticfield; and in the determination step, comparison is conducted using avalue obtained after removing an offset value included as a directcurrent component by extracting only a frequency component correspondingto the magnetic field from the output signal of a detector elementobtained in the measurement step.
 25. The method for measuring magneticmolecules according to claim 21, characterized in that the methodfurther comprises a binding acceleration step for generating a magneticfield for drawing the magnetic molecules close to the sensor surface bya magnetic field generation means at the time of introducing themagnetic molecules to the sensor surface.
 26. The method for measuringmagnetic molecules according to claim 25, characterized in that in thebinding acceleration step a magnetic field is applied of an intensitysuch that magnetization of the magnetic molecules becomes saturated, andin a step for acquiring the intensity of a magnetic field, a magneticfield is applied of an intensity such that magnetization of the magneticmolecules does not become saturated.
 27. The method for measuringmagnetic molecules according to claim 21, characterized in that themethod further comprises a stirring step for stirring magnetic moleculesby alternately producing magnetic fields, after introducing the magneticmolecules to the sensor surface, by means of a first magnetic fieldgeneration means arranged in a position facing the sensor surface and asecond magnetic field generation means arranged on the backside of thesensor surface.
 28. The method for measuring magnetic moleculesaccording to claim 27, characterized in that in the stirring step amagnetic field is applied of an intensity such that magnetization of themagnetic molecules becomes saturated, and in the step of acquiring theintensity of the magnetic field, a magnetic field is applied of anintensity such that magnetization of the magnetic molecules does notbecome saturated.
 29. A method for measuring objects to be measuredusing a biosensor according to claim 1, characterized in that moleculesthat bind specifically to the objects to be measured for binding areused as the magnetic molecules, the method comprising the steps of:determining an amount of the magnetic molecules bound specifically toobjects to be measured using a biosensor; and determining an amount ofobjects to be measured based on the amount of the magnetic molecules.30. A method for measuring objects to be measured using a biosensoraccording to claim 1, characterized in that molecules that arereversibly exchangeable for the objects to be measured for binding areused as the magnetic molecules, the method comprising the steps of:determining an amount of magnetic molecules bound in place of theobjects to e measured using a biosensor; and determining an amount ofobjects to be measured based on the amount of the magnetic molecules.